Campylobacter is a significant food borne pathogen in livestock products, including poultry. It is thus desirable to provide strategies for combating its deleterious effects in humans. Elimination of these pathogens from livestock can serve as a means to reduce the incidence of infection in humans and reduce disease in farm animals that are affected. Glycosylated proteins and in particular certain glycan moieties of these compounds represent viable targets for immunologic strategies to reduce or eliminate the presence of these organisms in livestock, in order to reduce the risk of human contamination from eating or handling animal products as well as contamination by fecal shedding of bacteria from livestock manure. Glycoproteins and their fragments such as glycan moieties may also provide a basis for vaccines and antibody preparation which target campylobacter infections.
It is also desirable to provide research tools having general application for studying protein glycosylation. Glycosylation of proteins was once considered to be specifically a eukaryotic phenomenon, but it is now clear that it is widespread in both the Archaea and Eubacteria domains (1,2). Glycosidic linkages of both the N- and O- types have been identified in a diverse group of prokaryotic organisms with a preponderance of N-linked sugars apparent in the Archaea while linkage units of the O-type predominate in glycoproteins identified thus far in the Eubacteria (1,2). In addition, bacterial N- and O- linkages are formed with a wider range of sugars than those observed in eukaryotic glycoproteins.
The present inventors have discovered that certain glycopeptides and gylcan moieties form an effective basis for vaccines and antibodies against multiple strains of campylobacter bacteria, by virtue of their ubiquitous presence on the surface of these bacteria in an invariant (or nearly invariant) form across multiple strains and species. This glycan is also present as a component of a plurality of surface glycoproteins of campylobacter thus enhancing its value as an immunologic target.
Recently a gene locus was identified in the enteric pathogen Campylobacter jejuni, which appears to be involved in the glycosylation of multiple proteins. This provided the first evidence of a pathway for wide-spread protein glycosylation in a gram-negative bacterium (3). Mutagenesis of genes within this locus, termed pgl (for protein glycosylation), resulted in loss of immunogenicity in multiple proteins. The glycan moieties of these proteins were also shown to be recognized by antisera from experimentally infected human volunteers (3). Removal of the glycan moieties by pgl mutation resulted in decreased adherence and invasion in vitro and loss of mouse colonization in vivo (4), suggesting that protein glycosylation influences the virulence properties of this organism.
The present inventors have identified and characterized post-translational modifications of proteins in C. jejuni strain NCTC 11168, the strain for which the whole genome sequence has been described by Parkhill et al. (6). Among the proteins giving rise to multiple spots on 2D gels was PEB3, or Cj0289c, a major antigenic protein of C. jejuni first described by Pei et al. (7). When purified and analysed by 1D SDS-PAGE, it revealed two bands with a mass difference of ˜1500 Da, both of which had N-terminal sequences corresponding to authentic PEB3. Concurrent with our observations on PEB3, Linton et al. (8) identified two putative glycoproteins from C. jejuni by use of the GalNAc-specific lectin, soybean agglutinin, one of which was PEB3, and the other a putative periplasmic protein Cj1670c, which they named CgpA. The authors also observed a number of other putative glycoproteins, based upon their ability to bind to the lectin, but these were not identified. Furthermore, protein binding to the lectin was also affected by mutagenesis of genes in the pgl locus. In addition, we have shown that mutation of a gene pglB, whose homology to the STT3 subunit of the N-linked oligosaccharyltransferase of Saccharomyces cerevisae suggested a role in glycoprotein biosynthesis (3,9), specifically affects the glycosylation of the identified glycoproteins.
Moreover, carbohydrates are implicated in a variety of functions in all domains of life. While in general there is high degree of variance of glycoproteins and their glycan moieties in antigenic surface glycans between bacterial strains and related species, this is not always the case and such exceptions present suitable excellent candidates for antibody or vaccine-based strategies for eliminating bacteria. In particular, one such carbohydrate is the glycan moiety of campylobacter glycoproteins. Our recent efforts to characterize the glycome of the important foodborne pathogen Campylobacter jejuni, has led to the elucidation of all surface glycan structures, namely, lipooligosaccharide (LOS), capsular polysaccharide (CPS), N-linked glycans, and O-linked glycans (5, 12-15)
It has been shown by the present inventors that the heptasaccharide, which is described in this specification, is common to at least several Campylobacter species and numerous strains including species that are important as human and veterinary pathogens, and is a component of multiple glycoproteins including Cj nos. 0114, 0200c, 0289c, 0367c and others. This glycan moiety is also strongly immunogenic and as such this glycan (and related fragements and glycopeptides) is a good candidate for use as a vaccine for active immunization against multiple strains and species of campylobacter and as the basis for antibodies or antibody fragments suitable for targeting of campylobacter in a human or in livestock.
As used herein “livestock” includes mammals and poultry.